The Bacillus thuringiensis Cry proteins are novel bacterial toxins with practical application as biological insecticides for the targeting of specific crop pests (caterpillars, beetles, nematodes) and disease vectors (mosquitoes). Insect ingestion of B. thuringiensis crystals [or spores and crystals] leads to solublization and proteolytic processing of Cry proteins that are thought to oligomerize and then insert into the midgut cells of insect. In the leading model for mechanism of action, this insertion results in the formation of cation-specific ion channels that collapse the electrochemical gradient of the midgut cells and ultimately leads to insect death. One important step towards understanding how Cry proteins insert into membranes would be the availability of Cry protein structures in both their soluble and membrane-inserted forms. Crystallographic efforts have provided structures of several Cry protein variants in their soluble forms, but as yet no structure of a Cry protein in its membrane inserted-form has been determined. This R21 grant will explore strategies towards the structural determination of such a membrane-inserted Cry toxin. PUBLIC HEALTH RELEVANCE: The Bacillus thuringiensis Cry proteins are novel bacterial toxins with practical application as biological insecticides for the specific targeting of insect crop pests and disease vectors. This proposal seeks to determine the structure of Cry toxins in the form that kills insects, knowledge that could be used to generate improved toxins. Such super Cry toxins could be used to improve human health by increasing crop yields, thereby helping to reduce human malnutrition, and by killing mosquitoes that can transmit diseases, resulting in lower incidence of sickness.